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*
Division of Clinical Immunology and Allergy and
Division of Infectious Diseases, Department of Medicine, University of California Medical School, Los Angeles, CA 90095; and
Pathophysiology Division, Lovelace Respiratory Research Institute, Albuquerque, NM 87185
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Abstract |
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2
generation and uncoupling of oxidative phosphorylation (decreased
intracellular ATP levels). N-Acetylcysteine reversed these
mitochondrial effects and ROS production. Overexpression of the
mitochondrial apoptosis regulator, Bcl-2, delayed but did not suppress
apoptosis. Taken together, these results suggest that DEP chemicals
induce apoptosis in macrophages via a toxic effect on
mitochondria. |
Introduction |
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Although the identity of DEP xenobiotics leading to acute lung injury are unknown, organic extracts made from DEP generate reactive oxygen species (ROS) in microsomes made from lung tissue (12). ROS are also involved in the toxic and proinflammatory effects of DEP in mice in vivo (6, 13, 14). ROS generation in microsomes was dependent on cytochrome P450 reductase activity, and the effects of the DEP chemicals in this assay can be inhibited by NaBH4, which reduces quinones to hydroxy derivatives (12). This suggests that quinones or oxidized polycyclic aromatic hydrocarbon (PAH) derivatives are responsible for ROS generation (12). Among the cell types that participate in ROS production in response to PM deposition in the lung is the macrophage (15, 16, 17, 18, 19). We are particularly interested in the role of macrophages in PM-induced inflammation for the following reasons: 1) macrophages are the principal cells that remove DEP and other PM from the bronchoalveolar region of the lung (20, 21); 2) macrophages phagocytose DEP, leading to the accumulation of toxic intracellular DEP concentrations (19); and 3) macrophages are endowed with enzymatic pathways for ROS generation, including inducible cytochrome P450-dependent pathways (22, 23, 24).
We have recently shown that phagocytosis of DEP leads to programmed cell death (PCD) in macrophages (19). The same response is induced by an organic DEP extract and is suppressed by a synthetic sulfhydryl (SH) antioxidant, suggesting that the oxidative effects of DEP chemicals are involved in apoptosis (19). A key question is what are the intracellular targets for DEP-induced ROS generation and cell death. Mitochondria have recently received a lot of attention for their involvement in apoptosis induction and apoptosis-related ROS generation (25, 26, 27, 28, 29, 30, 31, 32). These mitochondrial pertubations involve indirect or direct effects on the permeability transition (PT) pore (25, 26, 27, 28, 29). The PT pore is comprised of a complex assembly of inner- and outer-membrane proteins that regulate mitochondrial membrane permeability changes, ROS production, and the release of apoptogenic factors, such as cytochrome c (25, 26, 27, 28, 29, 30, 31, 32).
The aim of this study was to investigate whether organic chemicals
extracted from DEP induce PCD via a mitochondrially mediated pathway.
In addition, we wanted to assess the role of ROS in these events and
whether antioxidants and Bcl-2 can reverse the cytotoxic effects of DEP
chemicals. Our data demonstrate that macrophage exposure to a methanol
extract of DEP induces an orderly sequence of events in which the
mitochondrial membrane perturbation leads to a decrease in
mitochondrial membrane potential (
m),
cytochrome c release, structural damage to mitochondrial
inner membrane, O2 production, and uncoupling of oxidative
phosphorylation. These results indicate DEP toxicity involves a
mitochondrial pathway and ROS production.
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Materials and Methods |
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RPMI 1640 and FCS were purchased from Irvine Scientific (Santa Ana, CA). DMEM, penicillin-streptomycin, and L-glutamine were purchased from Life Technologies (Baltimore, MD). pUHD10–3-bcl-2 and pTRE-hygro (hygromycin resistance element) were generously provided by Dr. Sarah Cunningham (Clontech Laboratories, Palo Alto, CA). An E-Toxate kit, N-acetylcysteine (NAC), EDTA, propidium iodide (PI), pyrrolidine dithiocarbamate (PDTC), ascorbic acid, silibinin, luteolin, and zymosan were purchased from Sigma (St. Louis, MO). The annexin-FITC kit was purchased from Trevigen (Gaithersburg, MD). Hydroethidine (HE), 2',7'-dichlorofluorescein diacetate (DCF-DA), 3,3'-dihexyloxabarbocyanine iodide (DiOC6), 10 N-nonylacridine orange (NAO), and an ATP measurement kit were purchased from Molecular Probes (Eugene, OR). Anti-cytochrome c Abs were purchased from R&D Systems (Minneapolis, MN). Six-well plastic tissue-culture dishes were purchased from Fisher Scientific (Pittsburgh, PA). Monoclonal anti-Bcl-2 Ab was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Normal mouse serum was purchased from Atlanta Biologicals (Atlanta, GA). Bucillamine was provided by Keyston Biomedical (Los Angeles, CA).
Cell culture and stimulations
RAW 264.7 cells were cultured in a 5% CO2 atmosphere in DMEM containing 10% FCS, 5000 U/ml penicillin, 500 µg/ml streptomycin, and 2 mM L-glutamine. Rat pulmonary alveolar macrophages (PAM) were obtained from male Fischer rats by bronchoalveolar lavage as previously described (19). The cell yield was typically 5–7 x 106 cells/animal with >97% of the cells being macrophages. These cells were cultured in RPMI 1640 medium containing 2 mM L-glutamine, penicillin-streptomycin, and 10% FBS. For exposure to DEP extracts, aliquots of 3 x 106 cells were cultured in six-well plates in 3 ml of medium at 37°C for the indicated time periods. Some cultures received 20 mM NAC, 0.1–1 mM bucillamine, and 25–100 µg/ml each of PDTC, ascorbic acid, silibinin, or luteolin for 2 h before the addition of DEP extracts. Cells were harvested by scraping, washed three times in PBS, and then subjected to staining with fluorescent dyes.
Source and preparation of DEP extracts
DEP collected from a light-duty diesel source were obtained from Dr. Masura Sagai (National Institute of Environmental Studies, Tsukaba, Ibaraki, Japan) (6, 7, 12, 13). DEP extracts were prepared as previously described (19). Briefly, 100 mg of DEP were suspended in 50 ml of methanol and vigorously vortexed. The sample was sonicated for 2 min on ice and centrifuged for 10 min at 2500 rpm. Methanol extracts (1.5 ml) were aliquoted into a weighed Eppendorf tube. After methanol evaporation under nitrogen gas, the tubes were reweighed to determine the extract amount. The methanol-extractable component comprised 36% of the dry particle weight. The dried extracts were resuspended in DMSO to give the stock concentrations of 100 µg/µl. The stocks were stored in the dark at -20°C until use. DEP extracts were tested for endotoxin using the E-Toxate kit according to the manufacturer’s recommendation (Sigma). All of our extracts were essentially endotoxin free.
Cellular staining with fluorescent probes before performance of flow cytometry
Washed cells were stained with the fluorescent dyes at a
concentration of 106 cells/ml. Except for annexin
V and PI, the dyes were diluted in either DMEM or RPMI 1640. The
combinations and final concentrations of the dyes are as follows: 1) 1
µl of annexin V plus 10 µl PI in 500 µl binding buffer supplied
by the manufacturer (for assessment of early and late apoptotic events,
respectively; Ref. 19); 2) 20 nM
DiOC6 plus 2 µM HE (for assessment of
m and predominant O2 production,
respectively; Ref. 33); 3) 2.5 µM DCF-DA plus 2 µM HE
(for assessment of H2O2 and
O2 generation, respectively; Ref. 34); and 4) 100
nM NAO and 2 µM HE (assessment of cardiolipin mass and O2,
respectively; Ref. 35). Cells were incubated with these
dyes for 15 min (procedure 1) or 30 min (procedures 2–4) at 37°C in
the dark.
Flow cytometry
Flow cytometric analysis was performed using a FACScan (Becton Dickinson, Mountain View, CA) equipped with a single 488-nm argon laser. DiOC6, NAO, annexin V-FITC, and DCF were analyzed using excitation and emission settings of 488 nm and 535 nm (Fl-1 channel). PI was analyzed using excitation and emission settings of 488 nm and 575 nm (Fl-2 channel). HE was analyzed using excitation and emission settings of 518 nm and 605 nm (Fl-3 channel). Forward and side scatter were used to gate out cellular fragments.
ATP determination assay
Cells (3 x 106) were plated into 3.5-cm plates in 3 ml of media and rested for 4 h. Some cultures were preincubated with 20 mM NAC for 2 h. DEP extracts (100 µg/ml) were added to these cultures for varying amounts of time. Cells were collected, washed twice in PBS, and lysed in 200 µl H2O2. Samples were then boiled for 5 min and centrifuged at 14,000 rpm for 10 min. A Bradford assay was performed as previously described, and 500 ng protein was used to measure ATP content using a luciferin-luciferase ATP determination kit (Molecular Probes). The reagents were prepared according to the manufacturer’s recommendations and read in a Monolight 2010 luminometer. ATP concentration was calculated according to the manufacturer’s instructions
Western blotting analysis to assess mitochondrial release of cytochrome c
RAW 264.7 cells (3 x 106) were incubated together with 100 µg/ml DEP extract for the indicated time periods. Cells were lysed by three cycles of freeze-thawing in a lysis buffer containing 20 mM HEPES/KOH (pH 7.5), 1.5 mM MgCl2, 10 mM KCl, 1 mM NaEDTA, 1 mM EGTA, 1 mM DTT, 0.1 nM PMSF, and 250 mM sucrose. Cells were then spun in a Beckman Coulter Ultracentrifuge (Fullerton, CA) at 100,000 x g (45,000 rpm) at 4°C for 30 min. The supernatant (S-100 fraction) was collected and protein concentrations were measured by the Bradford method. One hundred micrograms of lysate was separated by 5–20% gradient SDS-PAGE and transferred to Immobilon-P membranes as previously described (19). Immunoblotting for cytochrome c was performed using a 1:500 dilution of the anti-cytochrome c Ab, followed by a 1:3000 dilution of HRP-coupled sheep anti-mouse Ab.
Transfection and generation of stable cell lines
A subclone of RAW 264.7, which stably expresses the tetracycline transactivator (tTA) protein (36), was a generous gift from Dr. Jawed Alam (Louisiana State University Medical Center, New Orleans, LA). RAW-tTA cells were transfected by electroporation using 20 µg of the tetracycline(tet)-repressible vector pUHD10–3 (31), into which human bcl-2(hbcl-2) was subcloned, together with 8 µg pTRE-hygro (hygromycin resistance element) as previously described (37). Cells were selected in 150 µg/ml hygromycin for 3 wk. Individual colonies were picked and transferred to tissue-culture dishes. Clones were then screened for the expression of hBcl-2 by immunoblotting. Eight of 10 clones expressed the hbcl-2 gene, irrespective of whether tet was added or omitted from the culture medium. Because this precluded us from comparing tet+ with tet- cells, we used RAW-tTA cells to compare the rate of apoptosis with that of RAW-Bcl2 cells during DEP exposure.
Preparation of opsonized zymosan
Opsonized zymosan particles were prepared as previously described (38). Briefly, zymosan was incubated with normal mouse serum (1 mg/ml) at 37°C for 30 min, followed by two washes with HBSS. The opsonized zymosan particles were then resuspended in HBSS at a final concentration of 500 mg/ml.
Western blotting analysis for hBcl-2
Cells (3 x 106) were grown in the presence and absence of 2 µg/ml tet for 24 h. Cells were lysed as previously described, and 100 µg lysate was separated by 12% SDS-PAGE and transferred to Immobilon-P membranes as described above. Immunoblotting for hBcl-2 was performed using a 1:1000 dilution of the anti-hBcl-2 Ab, followed by a 1:3000 dilution of HRP-coupled sheep anti-mouse Ab. To demonstrate that transfection with hBcl-2 increases total cellular Bcl-2 expression, we also performed Western blotting with a mAb that recognizes both the human and murine versions. This Ab was used at a 1:1000 dilution, followed by HRP-conjugated sheep anti-mouse secondary Ab at a dilution of 1:2000.
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Results |
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We have recently shown that DEP phagocytosis leads to apoptosis in
PAM and macrophage cell lines (19). In contrast, the
carbonaceous core that remains after methanol extraction of the
particle does not exert cytotoxic effects (19). This
suggests that the methanol-extractable component is responsible for
initiating PCD. We tested these methanol extracts in cellular apoptosis
assays. First, the extracts were dried, resuspended in DMSO, and then
added to rat PAM or the murine macrophage cell line RAW 264.7 for
18 h. Using dual annexin V/PI staining, we showed that the
methanol-extractable components induce early (annexin
V+/PI-) and late (annexin
V+/PI+) apoptotic changes
in primary as well as immortalized macrophages (Fig. 1
). Although the toxic effects of the DEP
extracts in both cell types were dose-dependent, there was a
reproducible trend toward a lower death rate in PAM (Fig. 1A) compared with RAW 264.7 cells at every test
concentration (Fig. 1B). This may reflect differences in
metabolic processing of DEP chemicals in these cells, or differences in
the abilities of these cells to defend themselves against oxidative
stress.
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C). Although
another synthetic SH compound, bucillamine, was also effective in
inhibiting DEP extract-induced cell death at 20- to 40-fold lower
concentrations than NAC, the flavanoids, silibinin and luteolin
(39, 40), were ineffective in preventing cell death (Table I). Moreover, other antioxidants such as
ascorbic acid (41) and PDTC (42) were also
ineffective in inhibiting cellular toxicity (Table I). This suggests
that the SH groups in NAC and bucillamine are responsible for the
inhibitory effects of these antioxidants.
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To determine the kinetics and types of ROS that are being produced
by DEP chemicals, we used a two-color flow cytometry approach, which
uses DCF-DA and HE (34). DCF-DA, which is freely cell
permeable, is trapped in the cell after the acetate moieties are
cleaved off and can be oxidized, mostly by
H2O2, to a green
fluorescent product. In contrast, HE is mostly oxidized by
O2 and forms ethidium bromide which emits red
fluorescence (34). After introducing RAW 264.7 cells to
the DEP extract, there was an immediate increase in DCF fluorescence,
which peaked after 2 h and then returned to baseline over the next
6 h (Fig. 2A). For HE,
there was a slow rise in the mean fluorescence intensity over the first
8 h, followed by a big rise in O2 production from that
point onwards (Fig. 2A). In a kinetic display of their mean
fluorescence intensities, it can be seen that there are two independent
phases of ROS production, namely an early phase of mostly
H2O2 production, followed
by a later phase of O2 production (Fig. 2B).
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A). Although
the DEP extract resulted in a sustained increase in DCF fluorescence
over a 4-h observation period, zymosan-treated cells responded with
H2O2 production for 1
h only (Fig. 3B). Although this suggests that the magnitude
or duration of ROS production may be a determinant for apoptosis, it is
also possible that a specific chemical event may be involved in
DEP-induced toxicity.
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The mitochondrial PT pore complex is regulated by numerous
effectors, including divalent cations, protons, the thiol redox state,
cross-linking of thiol groups, glutathione, ROS, lipid peroxidation,
and function of the Bcl-2 complex (30, 31, 32). Moreover,
redox-cycling chemicals and drugs also perturb mitochondrial function
(30). PT leads to the release of apoptogenic factors from
the intermembrane space. Immunoblotting of a mitochondrial-free
cytosolic fraction from RAW 264.7 cells showed the appearance of
cytochrome c in the cytosol (Fig. 4). The kinetics of cytochrome
c release, first seen around 4 h (Fig. 4), precedes the
onset of annexin V staining, which commences between 4 and 6 h
(19). Cytochrome c release in the cytosol leads
to caspase-9 activation in the presence of Apaf-1
(25, 26, 27, 28). This is compatible with our previous
demonstration that DEP exposure leads to caspase-9 activation in
macrophages (19).
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m, which can be assessed
with the fluorochrome DiOC6 (30).
Assessment of DiOC6 staining concurrent with HE
labeling showed an orderly sequence of events in DEP extract-treated
RAW 264.7 cells (Fig. 5A).
First, there was a decrease in DiOC6 staining,
beginning at 2 h (Fig. 5A). This drop in
m was progressive over the rest of the
observation period (Fig. 5A). Another consequence of PT was
the uncoupling of oxidative phosphorylation and interference in
one-electron transfers in the mitochondrial inner membrane
(32). This led to O2 production which was
detected by HE conversion (32, 43). In DEP extract-treated
cells, a prominent phase of O2 production started around
8 h, leading to the generation of
DiOC6low/HEhigh
and
DiOC6low/HElow
populations (Fig. 5A). The kinetics of O2
production in this experiment are compatible with the kinetics of
O2 production, shown in Fig. 2B. Although DEP
extracts have similar effects on m and
O2 production in PAM, the magnitude of these effects in PAM
was not as prominent as in RAW 264.7 cells (Fig. 5B). In
PAM, the DEP extract induced a single population of
DiOC6low cells, some of which
(26%) showed an increase in O2 production (Fig. 5B). This is consistent with the lesser toxicity of DEP
extracts in rat PAM (Fig. 1B).
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m is
linked to chemical-induced oxidative stress or thiol cross-linking
effects, we studied the effect of NAC on DiOC6/HE
fluorescence. Our data demonstrate that NAC effectively blocked the
m decrease as well as the increase in
O2 generation in RAW 264.7 cells (Fig. 5C). NAC had
similar protective effects in PAM (not shown). Although a redox-cycling
quinone, menadione, also induced a decrease in
m, which could be inhibited by NAC,
opsonized zymosan particles had no demonstrable effect on
m (not shown). Among the possible
explanations for the difference between the chemicals and zymosan is
that respiratory burst activity does not perturb mitochondrial
function, while chemical-induced oxidative stress does. Another
possibility is that redox-cycling chemicals induce PT by direct
complexing to pore proteins. In this regard, it is interesting that NAC
also protects mitochondria against dexamethasone-induced cell death
(32).
To study the effect of DEP chemicals on the structural integrity of the
mitochondrial inner membrane, we stained macrophages with a
fluorochrome, NAO, which binds stoichiometrically to an inner membrane
phospholipid, cardiolipin (35). After the introduction of
a DEP extract to RAW 264.7 cells, NAO fluorescence intensity decreased
precipitously from 
6 h onward (Fig. 6A). Moreover, the concurrent
measurement of O2 production (HE conversion), showed the
characteristic rise in ROS production from 8 h onward (Fig. 6A). Menadione had similar effects (not shown). Similar
observations were made in rat PAM, except that the magnitude of the
decrease in NAO staining and number of O-producing cells in the case of
PAM were lower than in RAW 264.7 cells (not shown).
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6 h onward (Fig. 6B). Moreover, previous incubation of
RAW 264.7 cells with NAC prevented the decline in ATP levels (Fig. 6B), showing that the impact of DEP chemicals on the
mitochondrium is either through ROS generation or a direct effect on
the PT pore. Bcl-2 overexpression causes a slight delay but does not protect against chemical-induced apoptosis
PT constitutes the first rate-limiting event of a common pathway
of apoptosis and can be induced either in a Bcl-2-regulated or in a
Bcl-2-independent fashion (32). In the case of
Bcl-2-regulated events, this mitochondrial protein prevents PT pore
opening as well as permeability changes in the inner and outer
membranes (43, 44, 45, 46). We asked whether Bcl-2 overexpression
can interfere with the proapoptotic effects of DEP chemicals in RAW
264.7 cells. This was accomplished by stable transfection of RAW-tTA
cells with a hBcl-2 construct using the tet-regulated
vector, pUHD10–3 (for details see Materials and Methods;
Ref. 36). Although tet was not effective in suppressing
Bcl-2 expression in stably selected RAW-hBcl2 cells (Fig. 7A), we were able to compare
these cells with RAW-tTA cells that contain a lesser amount of
endogenous Bcl-2 (Fig. 7B). Addition of a DEP extract to
RAW-tTA cells induced apoptotic (annexin
V+/PI-) changes in 24% of
the cells after 6 h (Fig. 7C). In contrast,
extract-treated hBcl-2 overexpressing cells showed only 2.7% annexin
V-positive cells at this point (Fig. 7C). However, by
10 h there was an advanced degree of apoptosis in
hBcl-2-overexpressing cells, indicating that DEP-induced toxicity is
largely a Bcl-2-independent process. In accordance with these findings,
Bcl-2 overexpression did not prevent a decrease in
m or cardiolipin degradation in
extract-treated cells (not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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m, followed by cytochrome c
release and development of membrane asymmetry (annexin V labeling).
Structural damage to the mitochondrial inner membrane, as evidenced by
a decreased cardiolipin mass, leads to a disruption in one-electron
transfer, O2 generation, and uncoupling of oxidative
phosphorylation. NAC prevented these mitochondrial changes, suggesting
that ROS are involved in the mitochondrial process or that NAC may
complex to the chemicals and/or the PT pore to prevent this damage.
Overexpression of the hbcl-2 oncogene delayed but did not prevent
apoptosis.
DEP are composed of a carbon core containing unburnt and adsorbed
petrochemicals (21). Methanol extracts made from DEP
induce ROS production in lung microsomes in a cytochrome P450
reductase-dependent manner (12). Although several lung
cell types may contribute to radical production, our results show that
PAM are able to increase
H2O2 and O2
production in response to exposure to organic DEP extracts (Fig. 1).
Uncoupling of electron transfer in the mitochondrium is a constant
feature accompanying apoptosis in a variety of different cell types
(32, 43). This likely is the source for the delayed
O2 production shown in Fig. 2. Although the source of
H2O2 production is
uncertain, this may be from a microsomally localized enzyme complex
involved in the metabolism or transformation of DEP chemicals
(12). It is also interesting that
H2O2 production by
zymosan-induced respiratory burst activity was associated with only a
small rise in cell death (Fig. 3). Although this observation may have
several explanations, two possibilities are that the magnitude and
duration of ROS production differ between these stimuli or that the ROS
produced by membrane-associated NADPH-oxidase do not impact the
mitochondrium.
Among the >400 chemicals that are present in DEP, PAH, nitro-derivatives of PAH, and oxygenated PAH derivatives (ketones, quinones, diones) are candidate chemicals that may contribute to ROS generation (8, 9, 10, 11). In this regard, Kumagai et al. (12) have shown that the oxidative effects of DEP extracts can be neutralized by NaBH4, arguing that quinones participate in ROS production in vitro. Quinones are reduced to semiquinone radicals by microsomally localized cytochrome P450 reductases (47). These semiquinone radicals produce ROS and can be reoxidized to the original quinone group, thereby initiating a futile redox cycle (47, 48). Therefore, it is interesting that menadione, a naphthoquinone that is readily reduced via NADPH-cytochrome P450 reductase to a semiquinone radical (49), had similar effects on the mitochondrium and cellular apoptosis as DEP chemicals (not shown). PAH may contribute to further ROS production during cytochrome P4501A1-dependent transformations. Characterization of the actual chemical groups responsible for ROS generation in macrophages needs to await the fractionation of DEP chemicals into functional groups. In addition to organic chemical compounds, the contribution of metal ions, such as Fe2+, should also be considered as a source of ROS generation (50).
ROS production by redox-cycling quinones or PAH may be involved in
DEP-induced apoptosis. ROS can induce lipid peroxidation or
cross-linking of thiol groups in proteins, both of which can trigger PT
(29, 32). Therefore, not surprisingly, mitochondria have
been implicated in the induction of apoptosis by a growing list of
pro-oxidative chemicals, including redox-cycling quinones and PAH
(29, 51, 52, 53). Although the data showing that NAC and
bucillamine interfere with DEP-induced apoptosis are compatible with
the involvement of oxidative stress (Fig. 1C and Table I),
there are other possible explanations for the role of these synthetic
SH compounds. One is that the reduced thiol groups in these
antioxidants may directly couple to quinones and the PT pore, thereby
preventing chemical damage to this pore. Because this covalent
interaction may also prevent the redox cycling of quinones, the
concomitant decrease in ROS production may appear to correlate with a
decreased rate of cell death. Failure of other classes of antioxidants
to protect against DEP-induced apoptosis favors the latter notion
(Table I).
Once perturbed by oxidative stress or covalently coupled DEP chemicals,
the PT pore initiates a sequence of events leading to apoptosis. The
first is a fall in m, detectable by reduced
labeling with DiOC6 (Fig. 5). This leads to or is
associated with the release of apoptogenic factors, including
cytochrome c (Fig. 4). Cytochrome c complexes
with Apaf-1, inducing it to associate with procaspase-9 and triggering
caspase-9 activation (27). This initiator caspase triggers
a further proteolytic cascade that ultimately induces cell death. In
this regard, we have previously demonstrated that DEP-induced apoptosis
is associated with procaspase-9 cleavage (19). Once
apoptosis is underway, mitochondria undergo further damages, including
structural damage to the inner membrane (Fig. 6A). This
leads to or is associated with defects in one-electron transfers and is
likely responsible for O2 production (Fig. 2A, 5,
A and B, and 6A). At this stage,
macrophages still have a normal size and normal dye exclusion (no PI
labeling), but may exhibit asymmetric phosphatidylserine distribution
in the membrane (detected by annexin V staining). This stage is
followed by cell shrinkage, apoptotic blebbing, and increased membrane
permeability (PI staining).
Intact mitochondria produce ATP. When PT involves a large fraction of
mitochondria in a cell, ATP may become depleted because of uncoupled
oxidative phosphorylation (Fig. 5B). This event may be
responsible for our finding that caspase inhibitors are only partially
effective in interfering with DEP-induced apoptosis (19).
If ATP depletion becomes severe, apoptosis may transition into
necrosis. This combined apoptosis-necrosis event is seen at higher DEP
extract concentrations (e.g., >100 µg/ml in RAW 264.7 cells).
Necrosis may augment the pulmonary effects of DEP because necrotic cell
fragments induce further inflammation.
PT constitutes the first rate-limiting event of the common pathway of
apoptosis and can be induced either in a Bcl-2-regulated or in
Bcl-2-independent fashion (32, 43). One way to distinguish
between those possibilities is through Bcl-2 overexpression, which
should interfere with the former but not the latter event. Although
overexpression of hBcl-2 somewhat retarded the rate of apoptosis (Fig. 7B), this was not effective in ultimately decreasing the
overall rate of cell death (Fig. 7C). Moreover, hBcl-2
overexpression did not prevent the extract-induced decrease in
m or NAO fluorescence in extract-treated
cells (not shown). This suggests that DEP-induced apoptosis is Bcl-2
independent.
Apoptosis may play an important role in the pulmonary toxicity of DEP. First, widespread apoptosis in PAM may interfere with lung defense against infectious organisms such as viruses. In this regard, it has been demonstrated that previous DEP exposure leads to increased mortality in mice infected with the influenza virus (54). DEP pre-exposure also interferes with the clearance of indicator particles from the airways of mice challenged via intratracheal instillation (55). Second, apoptosis of PAM may spread DEP chemicals via apoptotic bodies to neighboring tissue cells, thereby increasing tissue damage. In this way, phagocytosed apoptotic bodies may serve as a type of Trojan horse. Third, the mechanism of apoptosis in macrophages may also apply to neutrophils and other respiratory cell types. For instance, bronchial epithelial cells contribute to PM-induced respiratory inflammation and ROS production (56). Epithelial shedding, putatively as a consequence of apoptosis, may contribute to bronchial hyperreactivity in people with asthma. Fourth, macrophage apoptosis is associated with ROS production. Because many of these radicals are released into the extracellular environment, they may impact neighboring cells, which lead to their activation or apoptosis. Finally, an understanding of DEP toxicity will help to understand the toxicity of other inhalable particulates. An in vitro model of macrophage apoptosis may prove useful in characterizing the chemical compounds in other types of PM that induce acute respiratory effects.
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Acknowledgments |
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Footnotes |
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2 T.S.H. and N.L. contributed equally to this work.
3 Address correspondence and reprint requests to Dr. Andre E. Nel, Division of Clinical Immunology and Allergy, Department of Medicine, University of California, 10833 Le Conte Avenue, Los Angeles, CA 90095.
4 Abbreviations used in this paper: PM, particulate matter; DEP, diesel exhaust particles; DiOC6, 3,3'-dihexyloxabarbocyanine iodide; DCF-DA, 2',7'-dichlorofluorescein diacetate; HE, hydroethidine; NAC, N-acetylcystein; NAO, 10 N-nonylacridine orange; PI, propidium iodide; PAH, polycyclic aromatic hydrocarbon; PCD, programmed cell death; PT, permeability transition; PDTC, pyrrolidine dithiocarbamate; ROS, reactive oxygen species; tet, tetracycline; m, mitochondrial membrane potential; PAM, pulmonary alveolar macrophages; tTA, tet transactivator; hbcl-2, human bcl-2; SH, sulfhydryl.
Received for publication December 3, 1999. Accepted for publication June 14, 2000.
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 K. Donaldson, R. Aitken, L. Tran, V. Stone, R. Duffin, G. Forrest, and A. Alexander Carbon Nanotubes: A Review of Their Properties in Relation to Pulmonary Toxicology and Workplace Safety Toxicol. Sci., July 1, 2006; 92(1): 5 - 22. [Abstract] [Full Text] [PDF]
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 H. Bayram, K. Ito, R. Issa, M. Ito, M. Sukkar, and K. F. Chung Regulation of human lung epithelial cell numbers by diesel exhaust particles Eur. Respir. J., April 1, 2006; 27(4): 705 - 713. [Abstract] [Full Text] [PDF]
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I. Jaspers, J. M. Ciencewicki, W. Zhang, L. E. Brighton, J. L. Carson, M. A. Beck, and M. C. Madden Diesel Exhaust Enhances Influenza Virus Infections in Respiratory Epithelial Cells Toxicol. Sci., June 1, 2005; 85(2): 990 - 1002. [Abstract] [Full Text] [PDF]
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X. J. Yin, J. Y. C. Ma, J. M. Antonini, V. Castranova, and J. K. H. Ma Roles of Reactive Oxygen Species and Heme Oxygenase-1 in Modulation of Alveolar Macrophage-Mediated Pulmonary Immune Responses to Listeria monocytogenes by Diesel Exhaust Particles Toxicol. Sci., November 1, 2004; 82(1): 143 - 153. [Abstract] [Full Text] [PDF]
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 N. Li, J. Alam, M. I. Venkatesan, A. Eiguren-Fernandez, D. Schmitz, E. Di Stefano, N. Slaughter, E. Killeen, X. Wang, A. Huang, et al. Nrf2 Is a Key Transcription Factor That Regulates Antioxidant Defense in Macrophages and Epithelial Cells: Protecting against the Proinflammatory and Oxidizing Effects of Diesel Exhaust Chemicals J. Immunol., September 1, 2004; 173(5): 3467 - 3481. [Abstract] [Full Text] [PDF]
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 E. Beem, L. S. Holliday, and M. S. Segal The 1.4-MDa apoptosome is a critical intermediate in apoptosome maturation Am J Physiol Cell Physiol, September 1, 2004; 287(3): C664 - C672. [Abstract] [Full Text] [PDF]
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 N. Agopyan, J. Head, S. Yu, and S. A. Simon TRPV1 receptors mediate particulate matter-induced apoptosis Am J Physiol Lung Cell Mol Physiol, March 1, 2004; 286(3): L563 - L572. [Abstract] [Full Text] [PDF]
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 G. G. Xiao, M. Wang, N. Li, J. A. Loo, and A. E. Nel Use of Proteomics to Demonstrate a Hierarchical Oxidative Stress Response to Diesel Exhaust Particle Chemicals in a Macrophage Cell Line J. Biol. Chem., December 12, 2003; 278(50): 50781 - 50790. [Abstract] [Full Text] [PDF]
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 D. Upadhyay, V. Panduri, A. Ghio, and D. W. Kamp Particulate Matter Induces Alveolar Epithelial Cell DNA Damage and Apoptosis: Role of Free Radicals and the Mitochondria Am. J. Respir. Cell Mol. Biol., August 1, 2003; 29(2): 180 - 187. [Abstract] [Full Text] [PDF]
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 R Yanagisawa, H Takano, K Inoue, T Ichinose, K Sadakane, S Yoshino, K Yamaki, Y Kumagai, K Uchiyama, T Yoshikawa, et al. Enhancement of acute lung injury related to bacterial endotoxin by components of diesel exhaust particles Thorax, July 1, 2003; 58(7): 605 - 612. [Abstract] [Full Text] [PDF]
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 F M Ruemmele, S Schwartz, E G Seidman, S Dionne, E Levy, and M J Lentze Butyrate induced Caco-2 cell apoptosis is mediated via the mitochondrial pathway Gut, January 1, 2003; 52(1): 94 - 100. [Abstract] [Full Text] [PDF]
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Y.-C. T. Huang, J. Soukup, S. Harder, and S. Becker Mitochondrial oxidant production by a pollutant dust and NO-mediated apoptosis in human alveolar macrophage Am J Physiol Cell Physiol, January 1, 2003; 284(1): C24 - C32. [Abstract] [Full Text] [PDF]
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 E. Ferrero, D. Belloni, P. Contini, C. Foglieni, M. E. Ferrero, M. Fabbri, A. Poggi, and M. R. Zocchi Transendothelial migration leads to protection from starvation-induced apoptosis in CD34+CD14+ circulating precursors: evidence for PECAM-1 involvement through Akt/PKB activation Blood, January 1, 2003; 101(1): 186 - 193. [Abstract] [Full Text] [PDF]
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N. Li, M. Wang, T. D. Oberley, J. M. Sempf, and A. E. Nel Comparison of the Pro-Oxidative and Proinflammatory Effects of Organic Diesel Exhaust Particle Chemicals in Bronchial Epithelial Cells and Macrophages J. Immunol., October 15, 2002; 169(8): 4531 - 4541. [Abstract] [Full Text] [PDF]
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N. Rudra-Ganguly, S. T. Reddy, P. Korge, and H. R. Herschman Diesel Exhaust Particle Extracts and Associated Polycyclic Aromatic Hydrocarbons Inhibit Cox-2-dependent Prostaglandin Synthesis in Murine Macrophages and Fibroblasts J. Biol. Chem., October 11, 2002; 277(42): 39259 - 39265. [Abstract] [Full Text] [PDF]
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 F. Amersi, S. K. Nelson, X. D. Shen, H. Kato, J. Melinek, J. W. Kupiec-Weglinski, L. D. Horwitz, R. W. Busuttil, and M. A. Horwitz Bucillamine, a thiol antioxidant, prevents transplantation-associated reperfusion injury PNAS, June 25, 2002; 99(13): 8915 - 8920. [Abstract] [Full Text] [PDF]
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 S B Gordon and R C Read Macrophage defences against respiratory tract infections: The immunology of childhood respiratory infections Br. Med. Bull., March 1, 2002; 61(1): 45 - 61. [Abstract] [Full Text] [PDF]
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M. J. Whitekus, N. Li, M. Zhang, M. Wang, M. A. Horwitz, S. K. Nelson, L. D. Horwitz, N. Brechun, D. Diaz-Sanchez, and A. E. Nel Thiol Antioxidants Inhibit the Adjuvant Effects of Aerosolized Diesel Exhaust Particles in a Murine Model for Ovalbumin Sensitization J. Immunol., March 1, 2002; 168(5): 2560 - 2567. [Abstract] [Full Text] [PDF]
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M. S. Segal and E. Beem Effect of pH, ionic charge, and osmolality on cytochrome c-mediated caspase-3 activity Am J Physiol Cell Physiol, October 1, 2001; 281(4): C1196 - C1204. [Abstract] [Full Text] [PDF]
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 E. Fosslien Mitochondrial Medicine - Molecular Pathology of Defective Oxidative Phosphorylation Ann. Clin. Lab. Sci., January 1, 2001; 31(1): 25 - 67. [Abstract] [Full Text] [PDF]
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